Illuminating the dark space of neutral glycosphingolipidome by selective enrichment and profiling at multi-structural levels

Glycosphingolipids (GSLs) are essential components of cell membranes, particularly enriched in the nervous system. Altered molecular distributions of GSLs are increasingly associated with human diseases, emphasizing the significance of lipidomic profiling. Traditional GSL analysis methods are hampered by matrix effect from phospholipids and the difficulty in distinguishing structural isomers. Herein, we introduce a highly sensitive workflow that harnesses magnetic TiO2 nanoparticle-based selective enrichment, charge-tagging Paternò–Büchi reaction, and liquid chromatography-tandem mass spectrometry. This approach enables mapping over 300 distinct GSLs in brain tissues by defining sugar types, long chain bases, N-acyl chains, and the locations of desaturation and hydroxylation. Relative quantitation of GSLs across multiple structural levels provides evidence of dysregulated gene and protein expressions of FA2H and CerS2 in human glioma tissue. Based on the structural features of GSLs, our method accurately differentiates human glioma with/without isocitrate dehydrogenase genetic mutation, and normal brain tissue.

Additionally, "2OH" indicates that the position of the hydroxyl group is confirmed at carbon 2 in the N-acyl chain.

Target Name Primer
transmission electron microscopy.TiO2 MNPs were sent for Scanning Electron Microscopy (SEM) analysis.The SEM images and mapping of elements were acquired by Quanta 450 FEG Thermal Field Emission Scanning Electron Microscope.Nitrogen adsorption and desorption isotherms were measured using Quantachrome IQ.The Brunauer-Emmett-Teller (BET) method was utilized to calculate the specific surface areas (SBET) using adsorption data.By using the Barrett-Joyner-Halenda (BJH) model, the pore volumes and pore size distributions were derived from the adsorption branches of the isotherms.Zeta potential measurements were carried out on a Nano ZS90 zeta analyzer (Malvern Instruments Ltd.).The chemical compositions of TiO2 MNPs were detected through X-ray photoelectron spectra (XPS, Thermo ESCALAB 250Xi, Al Kα X-ray source).

Sample preparation
The diagnosis of isocitrate dehydrogenase genetic (IDH) mutation in human brain tissue samples was confirmed using Immunohistochemistry staining and Polymerase Chain Reaction Sequencing, specifically targeting Isocitrate Dehydrogenase 1 R132 and Isocitrate Dehydrogenase 2 R172.
Lipid extraction was performed based on a modified Folch method 5 .Briefly, 25 mg of human brain tissue sample was placed in a 10 mL-centrifuge tube. 1 mL of deionized water were added.The tissue sample was homogenized by a handheld homogenizer (Jingxin Technology) at 40,000 Hz for 5 min. 1 mL of methanol (MeOH) and 2 mL chloroform were further added and the tube was agitated for 10 min.The mixture was centrifuged at 12960 x g for 10 min and the bottom layer was extracted and transferred used above is 28 wt%, and the formic acid is 88 wt%.The separated GSL standards were reconstituted in 100 μL ACN, representing a 4-fold enrichment compared to the volume of the loading buffer.The same enrichment process was implemented with different concentrations of GSLs (12.5 nM, 500 nM, 1250 nM for each GSL standard).
To assess the recovery of GSLs under various concentrations, we also prepared a series of GSL standard mixtures by combing the five GSL standards at three different concentrations: 20 nM, 50 nM, and 5000 nM.Both the enriched GSLs and the GSL standard mixtures were then subjected to mass spectrometry (MS) analysis.For example, the recovery of GSLs at a 5 nM concentration was calculated by comparing the intensity of enriched GSLs (primarily at a 5 nM concentration with subsequent 4fold enrichment) to the standard GSL mixtures (with a concentration of 20 nM GSLs).
The recovery of sulfatides (at concentrations of 50 nM, 125 nM, 1250 nM) was also tested and analyzed using the same procedure above.
For the enrichment of GSLs from porcine brain lipid extracts, 100 μg of porcine brain lipids were mixed with 5 mg of TiO2 MNPs, 100 pmol of IS HexCer 18:1;O2/15:0, and 400 μL of loading buffer for 1 h agitation.TiO2 MNPs were separated by magnet and washed by washing buffer for two times.GSLs were released by 2 h agitation in eluting buffer.The separated GSLs were reconstituted in 100 μL of acetonitrile.
Pooled human brain lipid extracts were created by blending lipids from one IDH-mutant, one IDH-wildtype, and one normal sample in a 1:1:1 mass ratio.For GSL enrichment from human brain lipid extracts, an equivalent amount of lipids (either pooled or individual human brain lipid extracts) from 400 μg of human brain tissue was mixed S-24 with 5 mg of TiO2 MNPs, 200 pmol of IS HexCer 18:1;O2/15:0, and 400 μL of loading buffer for 1 h agitation.TiO2 MNPs were separated by magnet and washed by washing buffer for two times.GSLs were released by 2 h agitation in eluting buffer.The separated GSLs were reconstituted in 100 μL of acetonitrile (ACN).
The PB derivatization was performed using a homemade flow microreactor 7 .A GSL standard or ceramide standard at a concentration of 5 μM was dissolved in a 100 μL solvent (ACN/H2O, 5/1, v/v) containing 10 mM 2-acpy reagent.The solution was deoxygenated with nitrogen for 10 minutes and then injected into the flow microreactor for 15 seconds of UV irradiation (~254 nm).
FA extracts that were dried from a 50 μL stock solution and 10 mM PB reagent were dissolved in 100 μl ACN.The solution was then injected into the flow microreactor for 25 seconds of UV irradiation (∼254 nm).

RPLC conditions for GSLs
All LC separations were performed on a 20ADLC system (SHIMADZU, Tokyo, Japan) connected to an X500R QTOF mass spectrometer (SCIEX, Toronto, Canada).Intact The identified precursor ions were documented in the GSL library.Targeted MS/MS method was also used for C=C location analysis of unsaturated GSLs in porcine brain and pooled human brain samples.The mass spectra were recorded in the range from in Supplementary Table 3.
Analysis of total FA The LC method followed our previous report

Data analysis
Data analysis was performed using SCIEX OS (version 3.0.03339).GSL identification was conducted manually, considering RPLC retention time, accurate m/z, and fragmentation rules.All mass peaks with a signal-to-noise ratio (S/N) > 3 were utilized for identification.
The GSL species with relative ion abundances exceeded 1% of the most abundant GSL in MS 1 were chosen for relative quantitation at different structural levels.
Regarding %hFA, the selected GSLs should be commonly represented in 85% of individual human brain samples.Regarding %SPD, only those having %SPD higher than 5% were considered.For %C24, only those having %24 higher than 0.5% were considered.Given the high sensitivity of C=C location isomers, no additional criterium was used for their relative quantitation.

Protein extraction and Western blotting
For western blot and qRT-PCR analysis, we used samples from three normal tissues and three glioma tissues (two IDH-mutant and one IDH-wildtype).Protein extraction and quantification were performed on each 50 mg wet weight human brain tissue.The proteins were separated using SDS-PAGE (BIO-Rad, Hercules, CA, USA) and transferred to nitrocellulose membranes.These membranes were blocked using 5% non-fat milk and probed with the designated primary antibodies (diluted at 1:500-1000).
After incubation with horseradish peroxidase-conjugated secondary antibodies (diluted at 1:5000), the proteins were visualized through chemiluminescence, and the signals (BIO-Rad, Hercules, CA, USA) were quantified.

RNA extraction and qRT-PCR
Total RNAs were isolated using TRizol Reagent (Thermo Fisher Scientific) and reverse transcribed was proceeded with reverse transcribed kit superscript III (Invitrogen).The

Table 1 .
The table lists the amounts of two phospholipids removed from porcine brain through selective enrichment and the deviation in the amounts of two HexCers in porcine brain through selective enrichment with different batches of TiO2 MNPs.
and gas 2, 30 psi; declustering potential, 100 V.The SWATH method included an MS scan covering the range of m/z 100 to 500.Additionally, it included 23 MS/MS scans (m/z 100 -500) with various isolation windows (1 Da width, 0.1s accumulation time) and carried out with 3 technical replicates.The isolation windows in SWATH mode and collision energy are provided in Supplementary Data 7.